Pyruvate carboxylase (PC) is a biotin-dependent enzyme that catalyzes the bicarbonate and ATP- dependent carboxylation of pyruvate to oxaloacetate in a sequential reaction that takes place in two distinct active sites. Aberrant PC activity has been implicated in cancer, type-2 diabetes and aging-related diseases and, as such, PC represents a promising target for the development of novel chemical probes and therapeutics. To accomplish catalysis, a covalently attached biotin cofactor on the carrier domain of PC must translocate >50 between active sites located on physically separate polypeptide chains. This swinging domain mechanism is a feature of several enzymatic, multistep catalytic transformations, including those performed by pyruvate dehydrogenase, fatty acid synthase and polyketide synthases. Thus, PC serves as an accessible paradigm for a broad array of complex, modular enzymes that function via a swinging domain mechanism. In addition, PC is notably sensitive to an array of allosteric regulators, with acetyl-CoA serving as an allosteric activator and several carboxylic acids serving as allosteric inhibitors. Despite year of detailed kinetic studies and several recent X-ray structure determinations, the molecular basis for allosteric regulation and the driving force for carrier domain movement in PC remain unknown. This proposal outlines a series of structural, kinetic and biophysical approaches to study the effect of allosteric effectors on the structure and dynamics of PC. This project will significantly advance the molecular-level description of allosteric regulation and carrier domain movement in PC and will have implications to a broader subset of enzymes that function via a swinging domain mechanism. The proposed studies will focus on the unusual features of PC from Aspergillus nidulans, which exhibits a highly unusual response to allosteric activation. Our recently determined X-ray crystal structure of the apo A. nidulans PC enzyme serves to guide further structural and kinetic studies that will identify the source of its unusual allosteric response. This proposal also outlines new biophysical applications to directly observe the carrier domain of PC. Kinetic and biophysical methods are described to trace and trap the movement and location of this domain before and during catalytic turnover. These methods will be used to examine, for the first time, the interplay between allosteric regulation and carrier domain movement in PC. Together, these studies will significantly advance the description of allostery and dynamic domain motions in PC, a metabolically important enzyme that will serve as a valuable paradigm for the broader class of swinging domain enzymes.